Project Summary Studies from my laboratory during the past seventeen years have focused on the characterization of the molecular functions and biochemical properties of the Set1/MLL family of proteins. Their chimeras and mutations are associated with childhood leukemia and other forms of cancers. Our hope is that our molecular studies will advance our understanding of the molecular mechanisms of rearrangement and mutation-based cancer through this family of proteins. During the current funding cycle of this grant, my laboratory has employed genetics and biochemistry in multiple model systems including Drosophila and mammalian cell cultures. We demonstrated that Drosophila cells possess three Set1-related proteins: dSet1, Trithorax (Trx), and Trithorax-related (Trr), all found within COMPASS-like compositions capable of methylating histone H3K4. Mammalian cells possess two representatives for each of the three subclasses found in Drosophila for a total of six COMPASS family members: SET1A/SET1B (related to dSet1); MLL1 and MLL2 (related to Trx); and MLL3 and MLL4 (related to Trr). Furthermore, given that there is almost no sequence homology between many of the MLL translocation partners, for many years, it was unclear why MLL translocations into so many unrelated genes result in the pathogenesis of leukemia. Our biochemical studies on the purification of the MLL-chimeras demonstrated that many of the MLL translocation partners are part of the same macromolecular complex we named the Super Elongation Complex (SEC). We demonstrated that the translocations of MLL within any of the subunits of SEC result in the misrecruitment of SEC to the MLL target genes and in the perturbation of the transcriptional checkpoint control of these genes, triggering leukemic growth. Additionally, what we have learned is that recent cataloging of somatic mutations in cancer have identified a large number of mutations in the components of the MLL1-4 and Set1A/B complexes in both hematological malignancies and solid tumors. As a matter of fact, collectively MLL1-4 and Set1A/B appear to bear more mutations in different forms of cancers than p53. However, we know very little about the MLL1-4 and Set1A/B families in development and why their mutations are associated with cancer. Given that we have developed a fantastic set of reagents and tools within the past seventeen years towards these factors and their associated proteins in multiple model systems, my laboratory is in a very unique position to define the molecular bases of these factors' involvement in cancer pathogenesis for the purpose of targeted therapeutics. Therefore, the goals of this renewal application are the full molecular and biochemical characterization of MLL1-4 and Set1A-B and their complexes in the regulation of gene expression and development and how their mutations contribute to the pathogenesis of human cancer. The goals of this renewal application will be aggressively pursued via three specific aims. Specific Aim 1 is focused on the characterization of the molecular properties of the Trx/COMPASS family members (MLL1 and MLL2); identification of their molecular properties and specific recruitment to chromatin; and how their translocations contribute to leukemic pathogenesis. Specific Aim 2 will be focused in defining the role of the Trr/COMPASS family (MLL3 and MLL4) in enhancer monomethylation and how enhancer malfunction through specific mutations of the components of this family result cancer in pathogenesis. Specific Aim 3 is focused on the molecular characterization and structural studies of the Set1/COMPASS family (Set1A and Set1B) and how histone H3K4 trimethylation implemented by this class of enzymes is involved in the regulation of gene expression throughout development. We will take advantage of a variety of biochemical, molecular, and genetic tools in multiple model systems to address the proposed aims. These studies should (i) be instrumental for our understanding of the diverse roles of the COMPASS family in the regulation of the pattern of H3K4 methylation and how they regulate development and differentiation; and (ii) have a fundamental impact on our understanding of how mutations within the COMPASS family result in cancer. This information has the potential of proving helpful to investigators attempting to design rational approaches for the treatment of cancer.